The long term objectives of our studies are to understand the structure- function relationships of the Na,K-ATPase. Our studies to date have involved substituting various amino acids within the alpha subunit and evaluating the effect these replacements have on cation, ATP, and cardiac glycoside binding. The present grant represents a continuation of these studies as well as the development of a new expression system which will allow the effect of various amino acid substitutions on enzyme activity to be studied without interference with endogenous enzyme. During the upcoming grant period, we will evaluate the role of Asp 804 in cation transport, the only potentially transmembrane negatively charged amino acid that cannot be substituted without loss of activity. The other negatively charged amino acids when substituted individually do not severely affect enzymatic activity. We also plan to combine substitutions in these negatively charged residues to further explore their involvement in cation transport. In addition, we will follow up on our recent observation that serine 775 is important in K+ binding and investigate other oxy amino acids for their role in ion transport. Our studies of characterizing the ATP binding site using site directed mutagenesis will be continued. We also plan to develop a system which will make it possible to purify the enzyme for more detailed analysis such as cation occlusion. Our approach is to knockout both alpha subunit genes in a human cell line and introduce a rat alpha cDNA under the control of a regulatable promoter which will produce cell viability. Following introduction of a plasmid canning a substitution to be studied, the endogenous rescuable plasmid can be turned off with the only alpha subunit expressed will be the one whose properties are being studied. This will allow more detailed analysis to be carried out on our amino acid substitution enzyme such as cation occlusion. Studies of the binding site for cardiac glycosides will also be continued, using a comprehensive random mutagenesis approach to explore the entire molecule for those residues invoked in determining ouabain sensitivity. Finally, we will study the cardiac glycoside binding characteristics of human Na,K-ATPase carrying the alpha1, alpha2 and alpha3 isoforms.